3.1 Nervous system

KLFs play a crucial role in the development, function, and regeneration of the nervous system, responsible for regulating physiological processes such as neuronal differentiation, synaptic formation, and neuroprotective mechanisms. Specifically, KLF4 is known to be involved in the self-renewal and differentiation of neural stem cells within the nervous system,^{46, 47} as well as participate in nerve regeneration after injury, which facilitates remyelination and functional recovery after spinal cord injury via inducing astrocyte reprogramming.⁴⁸ KLF4 has been also identified as an effective inhibitor of axon growth in retinal ganglion cells (RGCs) and functions to repress axon and dendrite initiation and elongation by hippocampal neurons in vitro.⁴⁹ Additionally, KLF4 demonstrates a neuroprotective role through the regulation of the Nrf2/Trx1 pathway.⁵⁰

Both KLF6 and KLF7 have been shown to promote neurite growth.⁴⁹ A detailed investigation into KLF7 within the nervous system certified that knocking out KLF7 leads to impairments in axon growth and pathfinding in various areas such as the olfactory system, retina, and brain.⁵¹ Moreover, the capacity of KLF7 to boost the expression of Trk neurotrophin receptors might be crucial for the survival of RGCs following optic nerve injury.⁵² Therefore, KLF7 could not only accelerate neurite growth but also potentially enhance the responsiveness of neurons to trophic factors, ultimately improving neuroprotection postinjury.

KLF8 has been shown to regulate the expression of met and p53 crucial for the development of granule and purkinje cells,⁵³ as well as activate the Wnt/β-catenin signaling pathway, potentially impacting the progression of Alzheimer's disease.⁵⁴ KLF9 contributes to late-phase neuronal maturation in the developing dentate gyrus and during adult hippocampal neurogenesis,⁵⁵ which exerts various functions across different neuronal types.⁸ Furthermore, KLF15 acts as a transcriptional repressor of the rhodopsin and interphotoreceptor retinoid-binding protein (IRBP) promoters, as well as involved in restraining photoreceptor-specific gene expression in nonphotoreceptor cells.⁵⁶

3.2 Cardiovascular system

KLF2 has the potential to enhance endothelial nitric oxide synthase uncoupling through the Nrf2/HO-1 pathway in cases of endothelial injury, leading to improved cell viability, decreased lactate dehydrogenase (LDH) release, and a reduction in the oxidative stress response.⁵⁷ In endothelial cells, KLF2 suppresses prothrombotic factors like plasminogen activator inhibitor 1 and tissue factor, while simultaneously promoting the upregulation of the antithrombotic factor thrombomodulin (TM) in inflammatory conditions.^{4, 58}

KLF4 has been discovered to be expressed in various vascular cell types and contributes to the progression of vascular diseases by regulating the transcription of multiple genes through interactions with different partner proteins. Specially, KLF4 inhibits the expression of smooth muscle cell differentiation markers via associating with serum response factor, HDACs, and binding to p53. Furthermore, KLF4 collaborates with Runx2 to reinforce arterial medial calcification.⁵⁹

KLF5 has a variety of effects, such as promoting vascular smooth muscle cell proliferation, lipid deposition in the vessels, and inflammation, all of which substantially participate in atherosclerosis.^{60, 61} KLF5 could activate Cyclin D1 and suppress p21,⁶² and its interaction with poly-ADP-ribose-polymerase-1 obstructs the proapoptotic effect,⁶³ making a contribution to rescuing cells from undergoing apoptosis in the early phase and accelerating cell growth in the late phase to stimulate vascular remodeling in case of injury. Besides, KLF14 is also implicated in the regulation of diseases and pathological processes related to atherosclerosis.³ On the one hand, research has demonstrated that KLF14 plays a protective role by inhibiting lipoprotein lipase mediated by miR-27a, as well as diminishing the secretion of proinflammatory cytokines and lipid accumulation.⁶⁴ On the other hand, KLF14 suppresses the NF-κB signaling pathway by impeding the production of p65, thereby decreasing leukocyte adhesion to activated endothelial cells.⁶⁵

KLF13 is primarily expressed in the heart involved in the transcription network required for heart development.⁶⁶ which functions as a pivotal protective element that prevents cardiomyocytes from DNA damage and death.⁶⁷ Consistently, inhibition of KLF10 results in the alteration of Pten/Akt signaling to reduce cardiomyocyte apoptosis as well as enhance higher proliferation, playing a cardioprotective role in ischemic heart disease.⁶⁸ In patients with heart failure, KLF15 participates in the adapting response to heart pressure overload via regulating the expression of atrial natriuretic factor (ANP) and B-type natriuretic peptide (BNP).⁶⁹ Moreover, through interactions with factors including myocardin, TGF-β, myocyte enhancer factor 2, and GATA-binding protein 4,⁷⁰ KLF15 is able to suppress cardiac fibrosis and hypertrophy, ultimately consummating heart function upon myocardial remodeling processes triggered by mechanical or metabolic factors.⁷¹

3.3 Respiratory system

KLF4 performs distinct capabilities in the management of profibrotic mediators in various lung cell types. KLF4 in platelet-derived growth factor receptor (PDGFR)-β⁺-derived cells is of vital significance in the overload of myofibroblasts and extracellular matrix (ECM) generation and accumulation. Conversely, KLF4 in SMA⁺ cells exhibits a protective effect, and especially, downregulation of its expression could account for an increase in lung myofibroblasts.⁷² Furthermore, KLF4 in myeloid cells including macrophages and polymorphonuclear neutrophils serves as a crucial regulator of the early proinflammatory immune response. The stimulation of both cell types with S. pneumoniae dramatically boosts KLF4 expression, thus promoting a proinflammatory phenotype.⁷³

KLF6, as a downstream factor of LOX-1/TGF-β1 signaling pathway, is implicated in the pathological advancement of epithelial–mesenchymal transition (EMT)-mediated pulmonary fibrosis with diabetes.⁷⁴ Consistently, KLF15 also has the potential to counteract endoplasmic reticulum stress and inhibit excessive proliferation, migration, and ECM accumulation in lung fibroblast.⁷⁵ Besides, KLF7 has been uncovered to be highly expressed in lung adenocarcinoma tissues and associated with unsatisfied clinical outcomes.⁷⁶ KLF9 deficiency has been revealed to decrease the level of inflammatory factors and downregulate GSDMD expression, thereby alleviating lung injury and inflammatory responses.⁷⁷

3.4 Digestive system

KLF2 primarily exerts vasoprotective functions through activating target genes including endothelial nitric oxide synthase, TM, and c-type natriuretic peptide in liver sinusoid endothelial cells.⁷⁸ Nevertheless, KLF2 also upregulates CD36 expression via a binding site on its proximal promoter region, which partially contributes to liver steatosis.⁷⁹ KLF6 could mitigate the level of cellular oxidative stress and promote the responsibility for harmful stimuli. Further, with the modulation of Beclin1 transcription and activation of the mTOR/ULK1 pathway, KLF6 restrains excessive autophagy overactivation in order to safeguard the liver against ischemia/reperfusion injury.⁸⁰

Except for the parenchymal organs, the impacts of KLFs across the digestive tract deserve attention. It has been manifested that KLF4 ameliorates intestinal permeability, substantially enhances intestinal tight junction, and alleviates endotoxemia via repressing NF-κB transcription activity.⁸¹ KLF4, KLF5, KLF6, and KLF8 have been implicated in gastric carcinogenesis. Both KLF4 and KLF6 perform tumor suppressors.^{82, 83} KLF5 and KLF8 appear to promote the proliferation, invasion, and metastasis of human gastric carcinoma cells.^{84, 85} In colorectal tumors, KLF4, KLF6 and KLF9 have been identified to suppress colorectal carcinogenesis.^{86, 87} KLF4 restrains the expression of genes related to cell-cycle progression including CCND1 and ODC.⁸⁸ In contrast, KLF5 functions as colorectal carcinogenesis promoter.⁸⁹

3.5 Endocrine system

In the endocrine system, KLFs influence the biosynthesis, secretion, and function of various hormones by regulating gene expression, which in turn has a substantial impact on endocrine activity. KLF11 specifically controls the expression of the insulin genes by interacting with the insulin promoter and has been identified as a causal factor for maturity-onset diabetes of the young 7 (MODY7).⁹⁰ Besides, KLF11 functions as a dominant inhibitor of the caveolin-1 gene in response to cholesterol signals.⁹¹ KLF14 has also been implicated in the pathophysiological progress of metabolic diseases, such as obesity, insulin resistance, and T2D.⁹²

KLFs also play a crucial role in the differentiation of adipocytes and the metabolism of fatty acids. KLF3 orchestrates lipid metabolism via improving fatty acid β-oxidation (FAO).⁹³ KLF15 in adipose tissue modulates insulin exudation and resistance through diminishing stearoyl-CoA desaturase 1 and oxidative stress,⁹⁴ and it also has the capacity to shift fuel between glucose and fatty acids under different energy statuses in brown adipose tissue, which might enhance FAO accompanied by the promotion of the expression of Acox1 and Fatp1 while attenuating glucose oxidation.⁹⁵

3.6 Urinary system

KLFs are extensively expressed in tissues including kidney, bladder, and urethra, where they play crucial roles in the development, maintenance, and repair of the urinary system. These factors control genes associated with cell proliferation, differentiation, apoptosis, and inflammatory response, impacting both normal physiological conditions and diseases. KLF15 directly binds enhancers of regenerative genes such as adrenoreceptor alpha 1A to facilitate their expression in Xenopus laevis, the downregulation of which pharmacologically hinders nephric tubule regeneration, while the activation plays recuperating roles.⁹⁶

3.7 Reproductive system

Uterine and ovarian pathologies have been revealed to be both closely associated with the dysregulation of KLFs. Interestingly, higher levels of KLF12 were found in human endometrial cancer tissues compared with normal endometrium. The upregulation of KLF12 boosts cell proliferation and migration, as well as reins apoptosis through the activation of AKT signaling and promotion of CCND1 expression level, ultimately contributing to tumor growth.⁹⁷

Furthermore, multiple studies have discovered that KLF9, KLF11, KLF12, KLF15, and KLF16 are all involved in the occurrence and development of endometriosis.^{98, 99} Particularly, a notable reduction in KLF15 expression was observed in the mid-secretory epithelial endometrial cells of patients with endometriosis compared with individuals without the condition. KLF15, probably believed as a transcription factor for TWIST2, directly binds to its promoter regions to facilitate the process of EMT, orchestrating endometrial receptivity during embryo implantation.¹⁰⁰ KLF11 is abundantly expressed in reproductive tissues, where it alters endometrial metabolism by colocalizing with and recruiting the corepressor SIN3A/HDAC.¹⁰¹ Research has also shown that KLF11 not only hinders the development of endometriotic lesions but also effectively suppresses pathological scarring with Collagen1 repression.¹⁰² KLF9 knockdown may underlie progesterone resistance in endometriosis.¹⁰³ KLF12 restrains the decidualization of human endometrial stromal cells by inhibiting Nur77 expression.¹⁰⁴ Besides, KLF16 has been found to suppress endometrial CYP1A1 expression, which is associated with endometriosis and leads to decreased enzymatic activity¹⁰⁵ (Figure 2).

4.1 The direct modulatory effects of KLFs on regulating bone homeostasis

Current studies demonstrated that KLFs could directly affect the differentiation and activity of OBs and OCs by regulating certain gene expression and signaling pathways.

Certain KLFs are identified as critical regulators in OB-lineage cells.¹⁰⁶ KLF2 promotes the expression of the osteoblastic differentiation marker genes including Alp, Osx, and Ocn, as well as stimulates mineralization through upregulating Runx2 expression at both mRNA and protein levels.¹⁰⁷ Additionally, KLF10 also directly binds to regulatory elements located in the proximal region of the P1 promoter to activate Runx2 expression, as well as interact with Runx2 protein to increase the transcriptional activity.⁴⁵ Gao et al.¹⁰⁸ found that KLF5 promotes proliferation and osteogenesis differentiation of human periodontal ligament cells subjected to cyclic tensile stress through fibroblast growth factor (FGF2)-glycogen synthase kinase 3 beta (GSK-3β)/β-catenin signaling pathway. Similarly, Lin et al.¹⁰⁹ demonstrated that KLF4 promotes odontoblastic differentiation of dental papilla mesenchymal cells through the regulation of dentin matrix protein 1 (DMP1) and modulation of transforming growth factor-β (TGF-β) signaling pathways. Respectively, KLFs can also serve as downstream regulatory factors to participate in osteogenesis. It has been reported that interferon regulatory protein 2-binding protein 2 mediates the differentiation of OCs and OBs by targeting KLF2, thus affecting bone homeostasis.¹¹⁰ Yu et al.¹¹¹ found that conditional knockout of Cullin 4B (CUL4B) in mesenchymal stem cells (MSCs) can lead to impaired bone development along with low bone mass and reduced bone formation. Mechanistically, CUL4B suppresses KLF4 expression by directly binding to genes, epigenetically blocking the transcription, which promotes osteogenesis and inhibits adipogenesis of MSCs.¹¹¹ Interestingly, many members of the KLF family display the opposite role in osteogenic differentiation, inhibiting bone formation and mineralization. Kim et al.¹¹² found that KLF4 overexpression inhibits differentiation and mineralization of OBs. According to mechanistic studies, KLF4 acts as a negative regulator of Runx2, involved in the repression of the transcriptional process and DNA binding activity of Runx2 on target genes, including Alp and BSP.¹¹² Additionally, Weng et al.¹¹³ demonstrated that KLF14 downregulates Wnt3A expression by binding to the BTE2 locus in the gene promoter, abolishing Wnt3A mediated Wnt/β-catenin pathway activity and resulting in cell cycle arrest, which negatively regulates human bone marrow mesenchymal stem cells proliferation and osteogenic differentiation. Similarly, KLF15 elicited by glucocorticoid (GC) also inhibits bone formation in vivo by downregulating the Wnt signaling pathway, as well as upregulating peroxisome proliferator-activated receptor (PPAR)γ and FoxO3a.^{114, 115} KLF12-mediated inactivation of Wnt/β-catenin signaling is also involved in the inhibition of osteogenic differentiation.¹¹⁶

As a matter of fact, KLF2 is also involved in osteoclastic differentiation, promoting bone formation and attenuating bone resorption.^{117, 118} Mechanistically, KLF2 inhibits osteoclastic differentiation by downregulating the expression of OC-related genes such as c-Fos, NFATc1, and TRAP.¹¹⁰ Laha et al.¹¹⁹ showed that KLF2 downregulates Becn1 expression by reducing the levels of histone activation marks H4K8 and H3K9 acetylation in the promoter region of Becn1, leading to the alleviation of autophagy during osteoclastic differentiation. KLF4 is also involved in the negative regulation of OC differentiation. The overexpression of KLF4 in OBs results in the downregulation of RANKL expression via directly interacting with the RANKL promoter region to prevent the vitamin D receptor (VDR) from binding to the structure, which in turn diminishes the differentiation of OCs induced by 1,25(OH)₂D₃ in coculture of OBs and mouse bone marrow cells.¹¹² Additionally, Cicek et al.¹²⁰ found that KLF10 inhibits OC differentiation by suppressing the activation of NFATc1 pathway as well as repressing PKB and mitogen-activated protein kinase (MEK)/ERK signal transduction to reduce OC survival. On the contrary, Chen et al.¹²¹ discovered that KLF7 is capable of promoting the differentiation process by directly suppressing the expression of heme oxygenase-1 (HO-1), a negative regulator of OC differentiation. The phenomenon of the KLF family simultaneously regulating both osteoclastogenesis and osteoblastogenesis demonstrated a complex and balanced control required for maintaining bone homeostasis. This dual modulation reflected the multifunctional nature of KLF transcription factors, acting as pivotal nodes that integrate various signaling pathways to coordinate the actions of OCs and OBs. Such dual functionality maintained the exact modulation of bone resorption and formation processes, preventing pathological states including excessive bone destruction or formation. Dysregulation of KLF-mediated control can disrupt this balance, leading to conditions that either promote osteolytic or osteoblastic lesions. For instance, in prostate cancer (PCa), which typically results in osteoblastic bone metastases, upregulated KLFs can enhance OB activity, leading to abnormal bone formation and sclerosis. Conversely, in breast cancer, where osteolytic metastases are more common, altered KLFs can ramp up OC activity, leading to significant bone loss and the formation of destructive lesions. Overall, the ability of KLFs to concurrently influence both cell types demonstrates their potential as therapeutic targets in bone-related disorders.

Recent studies demonstrated that many members of KLF proteins participate in the regulation of cartilage homeostasis. KLF15 has been proven to take an active part in promoting chondrogenic differentiation of hMSCs through activating SOX-9 expression.¹²² KLF4 regulates membranous and endochondral ossification by precisely coordinating a series of cellular functions including the differentiation and migration of chondrocytes, OBs, and vascular endothelial cells.¹²³ Mechanistically, KLF4 overexpression accelerates type II collagen and SOX-9 expression and promotes sulfated-proteoglycan synthesis.¹²⁴ On the contrary, KLF5 overexpression reduces the expression of type II collagen and sulfated proteoglycan at the transcriptional and translational levels, leading to the dedifferentiation of chondrocytes.¹²⁵ Meanwhile, Shinoda et al.¹²⁶ demonstrated that mice with heterozygous deficiency (KLF5^+/−) exhibit impaired cartilage matrix degradation, delaying the replacement of cartilage with bone at the primary ossification center in the embryonic limbs. KLF5 induces transcriptional expression of MMP-9, leading to degradation of the cartilage matrix and ultimately contributing to endochondral ossification.¹²⁶ Dexamethasone, a type of GC drug, can induce apoptosis in growth plate chondrocytes and impair their differentiation, cell proliferation, and overall bone growth when used long-term. KLF2 has been demonstrated to promote proliferation and inhibit apoptosis of Dex-induced GPCs by targeting the Runx2-mediated PI3K/AKT and ERK signaling pathways.¹²⁷ All the above demonstrate that KLFs and downstream effector proteins construct a complicated transcriptional regulatory network that determines the differentiation and functions of OBs, OCs, and chondrocytes, collectively modulating skeletal development and homeostasis (Table 1).

4.2 The indirect effects of KLFs on bone homeostasis via energy metabolism

Over the last two decades, numerous studies have emphasized the crucial significance of KLFs in metabolic processes, which ultimately affect cellular physiological activities and the occurrence and development of diseases.^{134, 135} It is well known that bone is a highly metabolized tissue and needs to undergo metabolic reprogramming during bone remodeling to satisfy the high energy demand involved in many aspects including cell migration, cytoskeleton remodeling, differentiation, and secretion. ¹³⁶ Therefore, it can be inferred that KLF proteins potentially play significant roles in the regulation of bone remodeling by fine-tuning local metabolic programs to precisely match energetic demand, thereby modulating the dynamic balance of the bone matrix.

4.2.1 KLFs mediate bone homeostasis by regulating glycometabolism

Glycometabolism is widely recognized for its function as a primary energy source, encompassing three main metabolic pathways: oxidative phosphorylation (OXPHOS), glycolysis, and the pentose phosphate pathway.¹³⁷ To be specific, glucose is predominantly broken down through glycolysis and OXPHOS to produce ATP for energy provision. The pentose phosphate pathway supports cell growth by delivering the fundamental materials needed for macromolecule synthesis.¹³⁸ Accumulating evidence suggested that glycometabolism is indispensable for the activity and function of OBs and OCs. OBs are highly metabolically active cells that require substantial amounts of energy to synthesize the organic matrix of bone. Glycolysis is the main energy source of immature OBs, which stimulates the anabolic response of bone to parathyroid hormone and Wnt proteins including Wnt7B and Wnt3A in OB lineage cells.^139-142 Additionally, glucose transporter (GLUT) 1, GLUT3, and GLUT4 have been found to express in OBs.^143-145 GLUT1 is the primary glucose transporter in primary OBs, which promotes OB differentiation and bone formation by suppressing AMPK-dependent Runx2 degradation.¹⁴⁶ OCs, as the multinucleated cells responsible for bone resorption, are also dependent on glycolysis and OXPHOS for their energy demands. As OC differentiates, the number of mitochondria increases, accompanied by a higher OCR and elevated expression of glycolysis-related enzymes, such as hexokinase, phosphofructokinase, and pyruvate kinase. Additionally, the enzymes involved in the TCA cycle and OXPHOS are upregulated, indicating a surge in energy production.¹⁴⁷ Collectively, glucose metabolism regulates bone homeostasis by providing essential energy and intermediates for OB and OC function, thus influencing bone formation and resorption balance.

Recent studies demonstrated that KLF15 overexpression in adipocytes and myocytes promotes transcription of the insulin-sensitive GLUT, thereby facilitating the uptake of glucose into cells.¹⁴⁸ Furthermore, Yang et al.¹⁴⁹ demonstrated that KLF14 facilitates the phosphorylation of insulin receptor (InsR), which enhances insulin-mediated glucose uptake. The uptake of glucose can stimulate cells to produce ATP, which plays a crucial role in OB proliferation through increasing the expression of Alp, Runx2, and Osteocalcin during the initial phases of maturation in vitro.¹⁵⁰ This process is facilitated by elevated levels of intracellular Ca²⁺ signals, which activate the NFAT transcription factor during osteogenic differentiation. ATP enhances osteogenesis as indicated by Ca²⁺ deposition, which was consistent with the upregulation of OB genes such as BMP2, MMP-13, Col3a1, Ctsk, Flt1, and Bgn.¹⁵¹ Additionally, the vesicular release of ATP from human bone marrow stromal cells increases intracellular calcium concentration, in turn activating calcineurin and promoting the nuclear translocation of NFATc1, which directly orchestrates behavior of OBs and osteocytes.¹⁵² The ATP-dependent Ca²⁺ signals have the potential to enhance osteogenic differentiation protocols in tissue-engineered bone.

Studies have shown that in response to reactive oxygen species (ROS), cAMP response element–binding protein (CREB) induces the transcription of Ppargc1b that encodes for PPARγ coactivator 1 (PGC-1) during OC differentiation. Knockdown of Ppargc1 in vitro inhibits OC differentiation and mitochondria biogenesis, whereas deletion of the Ppargc1b gene leads to increased bone mass due to impaired OC function. Further studies have also observeddefects in PGC-1-deficient OBs, consistent with the aforementioned experimental findings both in vivo and in vitro. During OC development, the demand for iron is significant and is primarily met through the transferrin receptor 1 (TfR1), which facilitates OC differentiation by inducing mitochondrial respiration, increasing the production of ROS, and accelerating Ppargc1b transcription.¹⁵³ Myc has been evidenced to facilitate osteoclastogenesis via driving metabolic reprogramming during OC differentiation and functioning as a metabolic switch to an oxidative state, as well as being involved in the transcriptional induction of ERRα, a nuclear receptor that cooperates with the transcription factor NFATc1.¹⁵⁴ The Volker Ellenrieder group proved that KLF11 participates in TGF-β-mediated c-Myc gene expression in cancer, which proposes new ideas for bone tumors.¹⁵⁵

Additionally, the reprogramming of gluconeogenesis and anaerobic glucose metabolism mediated by KLFs alters the content of the energy molecule pyruvate. Previous studies have shown that pyruvate plays a crucial role in maintaining cell viability and survival of proliferative cultured calvarial OBs.¹⁵⁶ As an important intermediate product of glycometabolism, pyruvate could suppress hydrogen peroxide-induced generation of intracellular ROS in OBs, performing cytoprotective roles via a mechanism associated with the antioxidative property.¹⁵⁷ Additionally, Singh et al.¹⁵⁸ identified that stromal precursor cells deficient in Sod2 or intrinsically aged experience toxic accumulation of alpha-ketoglutarate due to impairment in pyruvate metabolism, which suppresses both osteogenic and adipogenic differentiation. With the deepening of research, the indirect regulatory pathway of KLFs affecting pyruvate content by altering anaerobic metabolism has gradually become clear in bone homeostasis, but its core mechanism still needs further confirmation (Figure 3).

5.1 DNA modification

DNA modification involves chemically altering DNA molecules to regulate gene expression and cellular function, which includes modifications like methylation, hydroxymethylation, and phosphorylation. During bone homeostasis, DNA modification impacts bone formation and absorption by controlling gene expression and signaling pathways in bone cells. Specifically, DNA methylation is intimately associated with cell differentiation and function regulation.¹⁸⁵ DNA methylation, a chemical modification process in which S-adenosylmethionine serving as a methyl donor covalently binds to specific bases on a DNA sequence under the catalysis of DNA methyltransferase (DNMT), is regarded as one of the most representative epigenetic modifications closely related to human development.¹⁸⁶ Previous studies have demonstrated that the alterations in promoter can impact the activity of OB and OC, disequilibrating osteogenesis and osteoclastogenesis.¹⁸⁷ It has been established that the methylation status contributes to determining cell lineage commitment. Typically, the methylation levels in promoter regions exhibit an inverse correlation with gene expression. Specifically, the promoter regions of osteogenic-related genes like Runx2 and osteocalcin are found to be completely hypomethylated in BMMSCs,¹⁸⁸ while the promoters of other osteogenic lineage-specific genes, such as ROR2, Dlx5, Runx2, and osterix, become demethylated during osteogenic differentiation, leading to increased gene expression in mRNA level.¹⁸⁹ Apart from its known function in osteogenic lineage differentiation, DNA methylation also plays a significant role in osteoclastogenesis. Studies have shown that OC formation is reduced in bone marrow macrophage precursor cells deficient in DNMT3a. Moreover, OC-specific DNMT3a knockout mice display high bone mass phenotypes and fewer osteoporotic possibilities compared with wild-type mice.¹⁹⁰

Recently, significant advances have been made in the effect of DNA methylation on KLF expression. It has been demonstrated that DNMT3B upregulation plays a role in mediating KLF5 hypermethylation induced by oxidative stress, which ultimately blocks osteogenic differentiation by reducing the expression and nuclear translocation of β-catenin.¹⁹¹ Yu et al.¹¹¹ demonstrated that CUL4B–RING E3 ligase (CRL4B) epigenetically represses KLF4 transcription such as DNA methylation and histone methylation through coordination with enhancer of zeste homolog 2 (EZH2), contributing to bone formation.

5.2 Histone modification

5.3 Noncoding RNA

5.3.3 Circular RNAs

Circular RNAs (circRNAs), derived from the process of back-splicing, are more stable than linear RNAs because of their unique loop structure, which is resistant to exonuclease-mediated degradation.^{218, 219} It has been demonstrated that circRNAs contain abundant miRNA binding sites and could function as competing endogenous RNAs to modulate the interaction between miRNA and target mRNA.^220-222 KLFs serve as downstream protein of miRNAs and their expression depends on the precise regulation of circRNA/miRNA interaction network. As mentioned above, KLF5 promotes cartilage matrix degradation by inducing MMP-9 expression at the transcriptional level.¹²⁶ Interestingly, the Kai Fu Wang group demonstrated that circ-ATRNL1 could directly target miR-153-3p to inhibit miR-153-3p-mediated KLF5 silencing, thereby ameliorating inflammatory responses, cell apoptosis, and ECM degradation.²²³ Similarly, circ-Strn3 could inhibit the matrix metabolism of chondrocytes in OA by competitively sponging miRNA-9-5p that targets KLF5²²⁴ (Figure 4 and Table 2).

TABLE 2. The coupling effect between KLFs and epigenetic regulation during bone remodeling.

Level	Modification	Key enzyme	Participants	Mechanism	References
Pretranscriptional modification	DNA methylation	DNMT3B	KLF5 gene	DNMT3B mediates KLF5 hypermethylation induced by oxidative stress, decreasing expression, and nuclear translocation of β-catenin to block osteogenic differentiation.	191
		EZH2	CRL4B/KLF4 gene	CRL4B epigenetically represses KLF4 transcription through DNA methylation coordination with EZH2.	111
	Histone methylation	Unclear	KLF2/ATG7 (H3K4)	KLF2 activates the ATG7 gene through the enrichment of methylation of H3K4.	198
	Histone acetylation	Unclear	KLF2/ATG7 (H3K27)	KLF2 activates the ATG7 gene through the enrichment of acetylation of H3K27.	198
		HAT (P300, PCAF)	KLF2/MMP-9 (H3K9 and H4K8)	KLF2 decreases the enrichment of active histone marks H3K9Ac and H4K8Ac and HAT (P300, PCAF) on the MMP-9 promoter region along with lower migration of activated monocytes to the rheumatoid arthritis sites.	201
		Unclear	KLF2/Becn1 (H4K8 and H3K9)	KLF2 decreases the level of histone activation marks H4K8 and H3K9 acetylation in the promoter region of Becn1 to inhibit the expression, thereby alleviating autophagy during osteoclastic differentiation.	119
		HDAC3 and P300	KLF4/DMP1 and Sp7	HDAC3 and P300 are recruited on the promoter region of KLF4 target genes DMP1 and Sp7, modulating the transcription to promote dentinogenesis and odontoblastic differentiation.	202
		HDAC	KLF11	KLF11 substantially suppresses CSC stemness of OS by restraining YAP/TEAD transcription activity via recruiting SIN3A/HDAC.	203
Posttranscriptional modification	MicroRNA		MiRNA-197-3p/KLF10	MiRNA-197-3p represses KLF10 expression to inhibit osteoblast differentiation.	209
			MiRNA-21-5p/KLF3	BMSCs-derived exosomal miRNA-21-5p inhibits KLF3 to enhance the proliferation and osteoblast differentiation.	225
			MiRNA-20-5p/KLF3	MiR-20a-5p promotes adipogenic differentiation through targeting KLF3.	210
			MiRNA-135a/KLF4	MiRNA-135a binds to 3′-UTR of KLF4 to inhibit its translation in OS cells.	226
			MiRNA-652/KLF9	MiRNA-652 binds to 3′-UTR of KLF9 to inhibit its translation, performing a carcinogenic role in OS cells.	227
			MiRNA-378/KLF9	MiR-378 downregulates the expression of KLF9, promoting the proliferation of OS cells.	228
			MiRNA-1236-3p/KLF8	MiRNA-1236-3p downregulates KLF8 to suppress the proliferative ability and induce apoptosis of OS cells.	211
			KLF5/miRNA‑487a/NKX3‑1	KLF5 directly binds to the promoter region of miR-487a to enhance its expression, which targets NKX3-1 to significantly facilitate the invasion and metastasis of OS cells.	229
			KLF8/miRNA‑429/SOX2	KLF8 directly binds the promoter region of miR-429 to inhibit its expression, which targets SOX2 to mediate cancer stem cell-like features in CD133+ OSCs.	230
	Long noncoding RNA		LncRNA Reg1cp/KLF3	Mutated lncRNA Reg1cp directly binds to KLF3 to inhibit its activity to promote angiogenesis during osteogenesis.	214
			LncRNA SNHG15/miRNA-7/KLF4	LncRNA SNHG15 sponge miRNA-7 to improve KLF4 expression to inhibit ECM degradation and alleviate OA progress.	215
			LncRNA MEG3/miRNA-9-5p/KLF4	LncRNA MEG3 sponge miRNA-9-5p to improve KLF4 expression, protecting chondrocytes.	216
			LncRNA LINC02381/miRNA-21/KLF12	LncRNA LINC02381 repressed osteogenic differentiation of human umbilical cord blood-derived MSCs through sponging miRNA-21 to enhance the inactivation of Wnt/β-catenin pathway mediated by KLF12.	116
			lnc-HLA-DQA1-5, lnc-RP11-127H5.1.1-1 and lnc-RTN2-1/miRNA-6799-5p, miRNA-1915-3p, miRNA-6764-5p, miRNA-6796-5p and miRNA-6895-3p/KLF2	lnc-HLA-DQA1-5, lnc-RP11-127H5.1.1-1 and lnc-RTN2-1 sponge miRNA-6799-5p, miRNA-1915-3p, miRNA-6764-5p, miRNA-6796-5p, and miRNA-6895-3p to regulate KLF2 in the mechanism of meniscus degeneration.	217
			LncRNA SNHG6/KLF2	LncRNA SNHG6 downregulates KLF2, accelerating OS progression.	231
			LncRNA KCNQ1OT1/miR-3666/KLF7	KCNQ1OT1 directly targets the miR-3666/ KLF7 axis and activates Wnt/β catenin signaling to facilitate OS progression.	232
	Circular RNA		CircRNA-LRP6/KLF2	CircRNA-LRP6 interacts with LSD1 and EZH2 to mediate their binding to the promoter regions of KLF2, inhibiting its expression and promoting OS development.	233
			CircATRNL1/miRNA-153-3p/KLF5	CircATRNL1 targets miRNA-153-3p to regulate KLF5 expression to protect against OA.	223
			CircCDK14/miRNA-1183/KLF5	CircCDK14 enhances KLF5 expression via targeting miR-1183, ameliorating interleukin-1β-induced chondrocyte damage in OA.	234
			CircStrn3/miRNA-9-5p/KLF5	CircStrn3 sponge miRNA-9-5p that targets KLF5 to inhibit matrix metabolism of chondrocytes.	224
			CircRNA_0078767/miRNA-889/KLF9	CircRNA_0078767 upregulates KLF9 expression by targeting miRNA-889, thereby inhibiting the progression of OS.	235

6.1 Osteoarthritis

OA is a prevalent joint disorder that affects people worldwide caused by the excessive production of cartilage MMPs, inflammatory response, and apoptosis, leading to the destruction of the cartilage matrix, inhibition of cartilage matrix synthesis,²³⁶ chronic pain, and disability.^{237, 238} Recent research demonstrated that multiple members of the KLF family are intimately involved in the onset or progression of OA.

Kawata et al.¹²⁸ identified that KLF4 and KLF2 are important modulators of chondrocytes. Adenoviruses containing KLF4 and KLF2 genes could significantly alleviate OA progression, including cartilage degradation, meniscus, and synovium inflammation. On the one hand, KLF4 and KLF2 are central transcription factors that directly bind to cartilage signature genes such as COL2A1, PRG4, and SOX to increase their expression. On the other hand, they are involved in protein kinase A (PKA)–RAP1–MEK–CREB signaling axis, ultimately suppressing mediators of inflammation and ECM-degrading enzymes.¹²⁸ Besides, KLF4 transcriptionally regulates InsR, which functions as a critical regulatory factor inactivating JAK2/STAT3 signaling, thus suppressing apoptosis of IL-1β-induced OA chondrocytes.¹³⁰ Another study also demonstrated that KLF4 promotes chondrocyte differentiation induced by simvastatin, which may be involved in the protective role of KLF4 in OA cartilage.¹²⁴ lncRNA MEG3 induces KLF4 expression by sponging miR-9-5p, thereby enhancing the protective role of KLF4 in chondrocytes.²¹⁶ Similarly, SNHG15 regulates the miR-7/KLF4/β-catenin axis, modulating ECM homeostasis to alleviate the progression of OA.²¹⁵ KLF2 has also been proven to block apoptosis of chondrocytes and matrix degradation by activating Nrf2/antioxidant-response element (ARE) signaling pathway.²³⁹

Sun et al.²⁴⁰ showed that early growth response 1 (EGR1), which is highly expressed in OA cartilage, activates KLF5 and β-catenin signaling to promote cartilage degeneration and hypertrophy. Interestingly, another study found that KLF5 is downregulated in OA cartilage tissues and protects chondrocytes against IL-1β-induced damage cell damage. The molecular mechanism suggested that circ-CDK14 could enhance KLF5 expression via targeting miR-1183, which serves as a negative upstream regulator of KLF5.²³⁴ Moreover, circStrn3 also inhibits matrix metabolism of chondrocytes in OA through competitively sponging miRNA-9-5p targeting KLF5.²²⁴ Circ-ATRNL1/miR-153-3p/KLF5 axis also plays a protective role in the development and progression of OA.²²³

MMP-3 is regarded as one of the most important cartilage-degrading enzymes that mediate the degradation process of type II collagen and aggrecan. The Yishuo Li group found KLF15 is downregulated in chondrocytes from OA patients, which may perform the protective role in OA chondrocytes. Mechanistically, KLF15 could bind to the promoter region of MMP-3 and inhibit its expression at the transcriptional level, thereby improving articular cartilage degradation in OA.¹³³ Additionally, KLF11 can suppress oxidative stress and apoptosis in OA chondrocytes by inhibiting the p38 MAPK signaling pathway.¹³² KLF10, which is a harmful factor overexpressed in both senescent chondrocytes and cartilage affected by OA,¹³¹ inhibits the proliferation and migration of chondrocytes via upregulation of Acvr1 and downregulation of Inhbb, significantly accelerating OA progression.²⁴¹ KLF10 deletion attenuates the tert-butyl hydroperoxide (TBHP)-induced senescence, blocking ROS production and maintaining mitochondrial homeostasis, thereby protecting cartilage against OA damage¹³¹ (Figure 5).

6.2 Osteoporosis

OP is a common orthopedic disorder, characterized by low bone mineral density (BMD), altered bone microstructure, and increased fracture incidence.^{242, 243} The imbalance between OB-mediated bone formation and OC-mediated bone resorption is the main contributor to OP.²⁴⁴ KLFs participate in the regulation of OB and OC activity.^{21, 245}

Several studies have demonstrated that KLF10 plays a crucial role in regulating OB differentiation, bone formation, and mineralization processes.^{45, 209} KLF10 could not only activate the transcription of the Runx2 gene via directly binding to the promoter and interacting with translated protein,⁴⁵ but also suppress osteoclastogenesis through the repression of the NFATc1 pathway and MEK/ERK signal transduction.¹²⁰ Further, according to quantitative computerized tomography studies, there is strong evidence that KLF10 is associated with volumetric cortical BMD.²⁴⁶ Compared with the normal population, the expression of KLF10 is significantly reduced in the skeletal tissues of osteoporotic patients.²⁴⁷

KLF4 is reported to modulate bone homeostasis by suppressing both OC and OB differentiation.¹¹² However, current studies demonstrated that KLF4 expression is time-specific and shows corresponding expression levels in different development stages, exerting diverse effects on bone mass. For instance, KLF4 conditional knockout in bone progenitor cells results in decreased OB production in mice, so as to significantly reduce bone mass,²⁴⁸ while mice with conditional deletion of KLF4 in OBs exhibit the opposite phenotype with high bone mass caused by the enhancement of bone formation.¹¹² Further studies indicated that tumor necrosis factor-α (TNF-α) significantly upregulates the expression of SOX5 transcriptionally, which inhibits the expression of OB markers such as Runx2, leading to a suppressive effect on the osteogenic differentiation of adult hMSCs, while KLF4 knockdown can reverse the inhibition.¹²⁹

The local vasculature actively participates in both bone formation and resorption by determining the fate of progenitor cells in the skeletal system. CD31^hiEMCN^hi vessels, located in the metaphysis and endosteum of postnatal long bones, have been proven to couple angiogenesis and osteogenesis.²⁴⁹ The Mi Yang group demonstrated that KLF3 could inhibit the expression of JunB and Vegfa, thus repressing the formation of the CD31^hiEmcn^hi endothelium in the bone marrow, ultimately leading to decreased bone mass.²¹⁴ Furthermore, Yang et al.¹¹⁵ demonstrated that GC treatment results in impaired bone formation by activating adipogenesis-associated KLF15/PPARγ2/FoxO3a/Wnt pathway and suppressing the canonical osteogenesis-related Wnt signaling. Additionally, KLF7 could directly bind to the promoter region of HO-1 to repress its expression, and thereby promote OC differentiation, ultimately facilitating OP progression.¹²¹

6.3 Osteosarcoma

Primary bone cancers, such as OS, Ewing sarcoma, and chondrosarcoma, are relatively rare but associated with high morbidity and mortality rates.^{250, 251} The development of bone cancers is partly ascribed to the metabolic changes caused by signal pathway reprogramming, which are vital to sustaining increased redox, bioenergetic, and biosynthesis demands of a tumor cell.²⁵² Recent studies showed that transcription factors play dominant roles in the initiation and advancement of OS, associated with major metabolic pathways.²⁵³

KLF2 has been shown to have growth-inhibitory, proapoptotic, and antiangiogenic effects, which are downregulated in malignancies.²⁵⁴ Zheng et al.²³³ found that circ-LRP6 is highly expressed in OS and interacts with LSD1 and EZH2 to mediate their binding to the promoter regions of KLF2, thereby inhibiting KLF2 expression and ultimately promoting OS development. Furthermore, KLF2 is downregulated by SNHG6, a novel type of molecule associated with the progression of multiple cancers, thereby accelerating OS progression.²³¹

KLF4 is a crucial regulator of normal cell proliferation and inhibits the proliferation of tumor cells, serving as a suppressor in many cancers. Qi et al.²⁵⁵ found that overexpression of KLF4 in OS cells led to the manifestation of traits commonly associated with OS CSCs including heightened sphere-forming potential, elevated levels of stemness-associated genes, and increased potential for metastasis. Contrarily, KLF4 knockdown could reduce colony formation in vitro and inhibit tumorigenesis in vivo, further confirming the oncogenic role in OS pathogenesis.²⁵⁵ A recent study identified that miR-135a is downregulated in OS tissue and inhibits cell invasion and expression by directly targeting KLF4.²²⁶ KLF6 serves as an important tumor suppressor gene frequently downregulated in multiple human cancers.²⁵⁶ Zhu et al.²⁵⁷ found KLF6 overexpression inhibits the viability, proliferation, and invasion of MG63 cells, and enhances cell apoptosis of the MG63 OS cell line via suppression of bcl-2 and MMP-9 and activation of p21.

MiRNAs serve as the upstream and downstream components of KLFs, widely participating in the occurrence and progression of OS. For instance, miR-652 negatively regulates KLF9 by directly interacting with its 3′-UTR, performing a carcinogenic role in OS cells.²²⁷ Similarly, MiR-378 also targets KLF9 to promote the cell proliferation of OS.²²⁸ Circ_0078767 has been demonstrated to enhance KLF9 expression by targeting miR-889, ultimately inhibiting OS progression.²³⁵ LncRNA KCNQ1OT1 is highly expressed in human OS tissues, and promotes OS cell proliferation, migration and invasion. Mechanistically, KCNQ1OT1 directly targets the miR-3666/KLF7 axis and activates Wnt/β-catenin signaling to facilitate OS progression.²³² In addition, it is demonstrated that miRNA-1236-3p suppresses the proliferative ability and induces apoptosis of OS cells by downregulating KLF8, an important cancer-promoting modulator.²¹¹ KLF8 directly binds the promoter region to inhibit the expression of miR-429, which directly targets SOX2 to mediate CSC-like features in CD133⁺ OS stem cell-like cells (OSCs).²³⁰ Besides, KLF5 enhances the expression of miR-487a by directly binding to its promoter region, which significantly facilitates the invasion and metastasis of OS cells via targeting NKX3-1²²⁹ (Figure 6).

6.4 Cancer-associated bone metastasis

According to research, bone metastasis is the third most common site for cancer metastasis, with around 70% of patients with metastatic prostate and breast cancer developing it.²⁵⁸ Bone metastasis is typically classified into three types: osteolytic, osteosclerotic, and mixed. Osteoblastic metastasis is typical for PCa, while breast cancer usually results in osteolytic metastasis.

Recently, increasing evidence indicated that several KLF members are involved in a variety of biological processes during breast cancer progression, including proliferation, migration, invasion, metastasis, and apoptosis.^{28, 259} Studies have demonstrated that increased expression of vascular cell adhesion molecule 1 in disseminated breast tumor cells plays a crucial role in attracting pre-OCs and facilitating their transformation into mature OCs, which ultimately leads to bone metastasis.²⁶⁰ Upon bone destruction, TGF-β is released from the bone matrix and signals to breast cancer, which may induce the expression of KLF10 and KLF11, which can further promote the development of bone metastasis.¹⁵⁵ Additionally, the Goutham Narla group demonstrated that KLF6-SV1, a splice variant of KLF6, has been found to be upregulated in hormone-refractory metastatic PCa. Its upregulation has been linked to the acceleration of PCa progression and metastasis through the modulation of apoptosis, cellular proliferation, and angiogenesis. PCa cells with overexpressed KLF6-SV1 metastasize more rapidly and disseminate to bone. Inhibition of KLF6-SV1 leads to spontaneous apoptosis in cultured PCa cell lines and also suppresses tumor growth in mice.²⁶¹

Indeed, bone homeostasis is a dynamic process governed by the balanced activity of OCs, which are responsible for bone resorption, and OBs, which are responsible for bone formation. Disruption of this balance is a hallmark of bone metastasis. Therefore, the regulation of bone metastasis by the KLF family may extend beyond their roles in cancer cell behavior, encompassing significant impacts on bone homeostasis, specifically osteoclastogenesis, and osteoblastogenesis. As mentioned above, various KLF members have been implicated in modulating the differentiation and activity of OCs. Dysregulation of these transcription factors can enhance OC formation, leading to excessive bone resorption. In the setting of cancer metastasis, such enhanced osteoclastic activity can create a favorable environment for cancer cells to invade and thrive within the bone matrix. On the other hand, KLFs also influence OB differentiation and function. Proper regulation of these pathways is essential for maintaining bone integrity. Abnormal expression of KLFs in OBs can impair bone formation, contributing to the imbalance between bone resorption and formation. In the context of osteoblastic metastasis seen in PCa, alterations in KLF expression could potentially enhance OB activity, leading to the characteristic bone lesions associated with this type of metastasis. By influencing both osteoclastogenesis and osteoblastogenesis, KLFs can create a bone microenvironment that either favors bone resorption or bone formation, depending on the type of metastatic cancer. This dual role highlights the importance of KLFs in maintaining skeletal homeostasis and the potential consequences of their dysregulation in the progression of bone metastases. Targeting the specific KLF-mediated pathways that regulate these processes could offer novel therapeutic strategies to mitigate bone metastasis in cancer patients.

6.5 KLFs act as the therapeutic target for treating bone destruction diseases

KLFs modulate bone homeostasis in multiple pathways including transcriptional regulation, energy metabolism as well as epigenetic activity. Aging, inflammation, stress, and other microenvironmental alterations affect the expression or activity of the KLF family, and thus lead to the disorder of downstream gene expression, ultimately causing a series of bone destruction diseases. Given the key role of KLFs in bone-related diseases (including OA, OP, OS, etc.), the development of drugs targeting KLFs may provide effective therapies for bone destruction diseases. For instance, Quercetin has been shown to decrease the expression of KLF4, which helps protect cells against oxidative damage and can potentially be used to treat oxidative-related diseases.²⁶² Furthermore, Li et al.²⁶³ reported that adriamycin treatment has been found to increase the expression of KLF4, leading to enhanced metastasis and cancer stemness in OS cells. Fortunately, simvastatin administration can markedly reverse adriamycin-induced tumorigenesis by downregulating KLF4 expression, providing potential new treatments for OS.²⁶³ Additionally, Kawata et al.²⁶⁴ identified a class I selective HDAC inhibitor moceinostat, which could improve tissue destruction and inflammation in OA by activating KLF4. Thiazolidinedione, a drug for treating type II diabetes, has been proven to KLF11 expression, which may be applied in the treatment of KLF11 silencing-mediated formation of OS CSCs.^{203, 265} These natural products and medicines possess certain curative effects with poor tissue specificity and promiscuity of binding. The regulation of gene expression mediated by miRNAs is owned to virtually all processes of skeleton development and has been shown its biological characteristics in bone destruction diseases, demonstrating the possibilities in the relevant treatment. MiRNA-based therapies targeting KLFs may provide significant clinical benefits to cancer patients potentially. For instance, BMMSCs-derived exosomes could enhance OB proliferation via miR-21-5p mediated inhibition of KLF3, which may be a potential therapeutic strategy to improve OP.²²⁵ Further research on the miRNA-KLF axis is required to fully understand the underlying mechanism, which has the potential to improve targeted therapies for bone destruction diseases.